Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions

Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein (LMP) 1, which is consistently expressed in multiple EBV-associated malignancies, has been proposed as a potential target antigen for any future vaccine designed to control these malignancies. However, the high degree of genetic variation in the LMP1 sequence has been considered a major impediment for its use as a potential immunotherapeutic target for the treatment of EBV-associated malignancies. In the present study, we have employed a highly efficient strategy, based on ex vivo functional assays, to conduct an extensive sequence-wide analysis of LMP1-specific T-cell responses in a large panel of healthy virus carriers of diverse ethnic origin and nasopharyngeal carcinoma patients. By comparing the frequencies of T cells specific for overlapping peptides spanning LMP1, we mapped a number of novel HLA class I- and class II-restricted LMP1 T-cell epitopes, including an epitope with dual HLA class I restriction. More importantly, extensive sequence analysis of LMP1 revealed that the majority of the T-cell epitopes were highly conserved in EBV isolates from Caucasian, Papua New Guinean, African, and Southeast Asian populations, while unique geographically constrained genetic variation was observed within one HLA A2 supertype-restricted epitope. These findings indicate that conserved LMP1 epitopes should be considered in designing epitope-based immunotherapeutic strategies against EBV-associated malignancies in different ethnic populations.

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.

Genetic variation in foliar carbon isotope composition in relation to tree growth and foliar nitrogen concentration in clones of the F-1 hybrid between slash pine and Caribbean pine

The objectives of this study were: (1) to quantify the genetic variation in foliar carbon isotope composition (delta(13)C) of 122 clones of ca. 4-year-old F-1 hybrids between slash pine (Pinus elliottii Engelm var. elliottii) and Caribbean pine (Pinus caribaea var. hondurensis Barr.,et Golf.) grown at two field experimental sites with different water and nitrogen availability in southeast Queensland, Australia, in relation to tree growth and foliar nitrogen concentration (N-mass); and (2) to assess the potential of using delta(13)C measurements, in the foliage materials collected from the clone hedges at nursery and the 4-year-old tree canopies in the field, as an indirect index of tree water use efficiency for selecting elite F-1 hybrid pine clones with improved tree growth. There were significant differences in foliar delta(13)C between the nursery hedges and the 4-year-old tree canopies in the field, between the summer and winter seasons, between the two experimental sites, and between the upper outer and lower outer canopy positions sampled. This indicates that delta(13)C measurements in the foliage materials are significantly influenced by the sampling techniques and environmental conditions. Significant differences in foliar delta(13)C, at the upper outer canopy in both field experiments in summer and winter, were detected between the clones, and between the female parents of the clones. Clone means of tree height at age ca. 3 years were positively related to those of the upper outer canopy delta(13)C at both experimental sites in winter, but only for the wetter site in summer. There were positive, linear relationships between clone means of canopy delta(13)C and those of canopy N-mass, indicating that canopy photosynthetic capacity might be an important factor regulating the clonal variation in canopy delta(13)C. Significant correlations were found between clone means of canopy delta(13)C at both experimental sites in summer and winter, and between those at the upper outer and lower outer canopy positions. Mean clone delta(13)C for the nursery hedges was only positively related to mean clone stem diameter at 1.3 m height at age 3 years on the wetter site. The clone by site interaction for foliar delta(13)C at the upper outer canopy was significant only in summer. Overall, the relatively high genetic variance components for foliar delta(13)C and significant, positive correlations between clone means of foliar delta(13)C and tree growth have highlighted the potential of using foliar delta(13)C measurements for assisting in selection of the elite F-1 hybrid pine clones with improved tree growth. (C) 2002 Elsevier Science B.V. All rights reserved.

The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics

Trans-membrane proteins of the p24 family are abundant, oligomeric proteins predominantly found in cis-Golgi membranes. They are not easily studied in vivo and their functions are controversial. We found that p25 can be targeted to the plasma membrane after inactivation of its canonical KKXX motif (KK to SS, p25SS), and that p25SS causes the co-transport of other p24 proteins beyond the Golgi complex, indicating that wild-type p25 plays a crucial role in retaining p24 proteins in cis-Golgi membranes. We then made use of these observations to study the intrinsic properties of these proteins, when present in a different membrane context. At the cell surface, the p25SS mutant segregates away from both the transferrin receptor and markers of lipid rafts, which are enriched in cholesterol and glycosphingolipids. This suggests that p25SS localizes to, or contributes to form, specialized membrane domains, presumably corresponding to oligomers of p25SS and other p24 proteins. Once at the cell surface, p25SS is endocytosed, together with other p24 proteins, and eventually accumulates in late endosomes, where it remains confined to well-defined membrane regions visible by electron microscopy. We find that this p25SS accumulation causes a concomitant accumulation of cholesterol in late endosomes, and an inhibition of their motility - two processes that are functionally linked. Yet, the p25SS-rich regions themselves seem to-exclude not only Lamp1 but also accumulated cholesterol. One may envision that p25SS accumulation, by excluding cholesterol from oligomers, eventually overloads neighboring late endosomal membranes with cholesterol beyond their capacity (see Discussion). In any case, our data show that p25 and presumably other p24 proteins are endowed with the intrinsic capacity to form highly specialized domains that control membrane composition and dynamics. We propose that p25 and other p24 proteins control the fidelity of membrane transport by maintaining cholesterol-poor membranes in the Golgi complex.

Vps20p and Vta1p interact with Vps4p and function in multivesicular body sorting and endosomal transport in Saccharomyces cerevisiae

Vps4p (End13p) is an AAA-family ATPase that functions in membrane transport through endosomes, sorting of soluble vacuolar proteins to the vacuole, and multivesicular body (MVB) sorting of membrane proteins to the vacuole lumen. In a yeast two-hybrid screen with Vps4p as bait we isolated VPS20 (YMR077c) and the novel open reading frame YLA181c, for which the name VTA1 has recently been assigned (Saccharomyces Genome Database). Vps4p directly binds Vps20p and Vta1p in vitro and binding is not dependent on ATP-conversely, Vps4p binding to Vps20p is partially sensitive to ATP hydrolysis. Both ATP binding [Vps4p-(K179A)] and ATP hydrolysis [Vps4p-(E233Q)] mutant proteins exhibit enhanced binding to Vps20p and Vta1p in vitro. The Vps4p-Vps20p interaction involves the coiled-coil domain of each protein, whereas the Vps4p-Vta1p interaction involves the (non-coiled-coil) C-terminus of each protein. Deletion of either VPS20 (vps20Delta) or VTA1 (vta1Delta) leads to similar class E Vps(-) phenotypes resembling those of vps4Delta, including carboxypeptidase Y (CPY) secretion, a block in ubiquitin-dependent MVB sorting, and a delay in both post-internalisation endocytic transport and biosynthetic transport to the vacuole. The vacuole resident membrane protein Sna3p (whose MVB sorting is ubiquitin-independent) does not appear to exit the class E compartment or reach the vacuole in cells lacking Vps20p, Vta1p or Vps4p, in contrast to other proteins whose delivery to the vacuole is only delayed. We propose that Vps20p and Vta1p regulate Vps4p function in vivo.

Contextual binding of p120ctn to E-cadherin at the basolateral plasma membrane in polarized epithelia

E-cadherin-catenin complexes mediate cell-cell adhesion on the basolateral membrane of epithelial cells. The cytoplasmic tail of E-cadherin supports multiple protein interactions, including binding of beta-catenin at the C terminus and of p120(ctn) to the juxtamembrane domain. The temporal assembly and polarized trafficking of the complex or its individual components to the basolateral membrane are not fully understood. In Madin-Darby canine kidney cells at steady state and after treatment with cycloheximide or temperature blocks, E-cadherin and beta-catenin localized to the Golgi complex, but p120ctn was found only at the basolateral plasma membrane. We previously identified a dileucine sorting motif (Leu(586)-Leu(587), termed S1) in the juxtamembrane domain of E-cadherin and now show that it is required to target full-length E-cadherin to the basolateral membrane. Removal of S1 resulted in missorting of E-cadherin mutants (EcadDeltaS1) to the apical membrane; beta-catenin was simultaneously missorted and appeared at the apical membrane. p120(ctn) was not mistargeted with EcadDeltaS1, but could be recruited to the E-cadherin-catenin complex only at the basolateral membrane. These findings help define the temporal assembly and sorting of the E-cadherin-catenin complex and show that membrane recruitment of p120(ctn) in polarized cells is contextual and confined to the basolateral membrane.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response [1, 2]. TNFalpha plays a key role in inflammatory disease [3]; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types [4, 5] was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

GAIP participates in budding of membrane carriers at the trans-Golgi network

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans -Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

Preserved xenogenic amniotic membrane as a patch on the repair of superficial corneal ulcers in rabbits

The aim of this study was to evaluate the effects of canine amniotic membrane, previously preserved in glycerin, used as a patch on the repair of experimentally-made superficial corneal ulcers and to compare corneal epithelization between the treated and non-treated groups. Xenogeneic amniotic membranes were collected aseptically and preserved in 99% glycerin at room temperature. Each animal was anesthetized and submitted to superficial corneal keratectomy of the left eye. The treated group received a fragment of canine amniotic membrane as a patch, while the control group had no treatment. The treated group showed blepharospasm, ocular discharge and conjunctival congestion. The membrane accelerated corneal repair in the beginning of the process, however, it delayed its conclusion (p<0.05). Treated eyes showed greater vessel formation and decreased corneal transparency (p<0.05). The stroma of the control group was thicker than that of the treated group (p<0.05). We suggest that amniotic membrane used in this manner can be applied as a therapy for superficial corneal ulcers in the beginning phases of the repair process.

CAUSE-FIT, POSITIVE ATTITUDES AND BEHAVIORS WITHIN HYBRID COLOMBIAN ORGANIZATIONS

ABSTRACTSocially oriented ventures have provided livelihoods and social recognition to disadvantaged communities in different corners of the world. In some cases, these ventures are the result of Corporate Social Responsibility (CSR) programs. In Latin America, this type of undertaking has responded positively to unmet social needs. The social cause drives these organizations and their human resources and they give high value to organizational cause-fit. This paper presents empirical evidence of the effects of perceived cause-fit on several worker attitudes and behaviors. Psychological contract theory was adopted as theoretical background. Employees working in a hybrid (for-profit/socially oriented) Colombian organization created by a CSR program participated in the survey. Data provided by 218 employees were analyzed using PLS structural equation modeling. The results suggest the ideological components of the employee-employer relationship predict positive attitudes and cooperative organizational behaviors towards hybrid organizations.

A hybrid PSO-ANFIS approach for short-term wind power prediction in Portugal

The increased integration of wind power into the electric grid, as nowadays occurs in Portugal, poses new challenges due to its intermittency and volatility. Wind power prediction plays a key role in tackling these challenges. The contribution of this paper is to propose a new hybrid approach, combining particle swarm optimization and adaptive-network-based fuzzy inference system, for short-term wind power prediction in Portugal. Significant improvements regarding forecasting accuracy are attainable using the proposed approach, in comparison with the results obtained with five other approaches.

A hybrid approach to multi-agent decision-making

In the aftermath of a large-scale disaster, agents' decisions derive from self-interested (e.g. survival), common-good (e.g. victims' rescue) and teamwork (e.g. fire extinction) motivations. However, current decision-theoretic models are either purely individual or purely collective and find it difficult to deal with motivational attitudes; on the other hand, mental-state based models find it difficult to deal with uncertainty. We propose a hybrid, CvI-JI, approach that combines: i) collective 'versus' individual (CvI) decisions, founded on the Markov decision process (MDP) quantitative evaluation of joint-actions, and ii)joint-intentions (JI) formulation of teamwork, founded on the belief-desire-intention (BDI) architecture of general mental-state based reasoning. The CvI-JI evaluation explores the performance's improvement

Short-term electricity prices forecasting in a competitive market by a hybrid PSO-ANFIS approach

In this paper, a novel hybrid approach is proposed for electricity prices forecasting in a competitive market, considering a time horizon of 1 week. The proposed approach is based on the combination of particle swarm optimization and adaptive-network based fuzzy inference system. Results from a case study based on the electricity market of mainland Spain are presented. A thorough comparison is carried out, taking into account the results of previous publications, to demonstrate its effectiveness regarding forecasting accuracy and computation time. Finally, conclusions are duly drawn. (C) 2012 Elsevier Ltd. All rights reserved.

Hybrid Wavelet-PSO-ANFIS Approach for Short-Term Electricity Prices Forecasting

A novel hybrid approach, combining wavelet transform, particle swarm optimization, and adaptive-network-based fuzzy inference system, is proposed in this paper for short-term electricity prices forecasting in a competitive market. Results from a case study based on the electricity market of mainland Spain are presented. A thorough comparison is carried out, taking into account the results of previous publications. Finally, conclusions are duly drawn.